Aav vectors for vascular gene therapy in coronary heart disease and peripheral ischaemia

ABSTRACT

The invention relates to the provision of a gene therapy for coronary heart disease and peripheral ischemia in mammals. One embodiment is an adeno-associated viral vector (AAV vector) comprising a first gene encoding a myocardin-related transcription factor A (MRTF-A). The invention further also relates to a pharmaceutical composition comprising an AAV vector of the invention and a pharmaceutically acceptable carrier. Methods for preparing the vector of the invention are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. §371 ofInternational Patent Application PCT/EP2015/057987, filed Apr. 13, 2015,published as International Patent Publication WO 2015/158667 on Oct. 22,2015, which claims the benefit of German Patent Application DE 10 2014207 153.4, filed on Apr. 14, 2014; a, the contents of all are herebyincorporated by reference.

FIELD OF THE INVENTION

The invention is the field of gene therapy. In particular, the inventionis directed to providing gene therapy for coronary heart disease andperipheral ischemia in mammals.

BACKGROUND OF THE INVENTION

In industrialized countries, coronary heart disease remains the mostcommon cause of death, in spite of improved treatments such asrevascularization of an occluded coronary vessel (Lloyd-Jones et al.,Circulation 2010, 121:e46-e215). Besides the manifestation of coronaryheart disease as an acute heart attack, myocardial ischemia may occurthrough a slow, chronic occlusion of a coronary vessel, which canprogress to heart insufficiency and even to cardiac failure (Suero etal., J Am Coll Cardiol 2001, 38:409-14).

Chronic ischemic disease of the heart or peripheral muscle is presentlytreated using surgical or interventional measures in order torevascularize constricted or occluded vascular networks. Although drugtherapy following the re-opening of an occluded vessel, and thusevent-free survival of patients, has been greatly improved in the lastyears, a number of patients still develop heart insufficiency (Levy etal., N Engl J Med 2002, 347:1397-402). In a growing population ofpatients, conventional therapeutic strategies become exhausted andclinical benefit is then expected from adjuvant neovascularizationtherapies (angiogenesis/arteriogenesis).

Previous pre-clinical (Kupatt et al., J Am Coll Cardiol 2010, 56:414-22)and clinical studies (Rissanen and Ylä-Herttuala, Mol Ther 2007,15:1233-47) failed to reveal any increase in perfusion, if angiogenesis(capillary growth) was reinforced in the absence of microvesselmaturation, i.e. recruiting of pericytes and smooth muscle cells (Jain,Nat Med 2003, 9:685-693; Potente et al., Cell 2011, 146:873-887).Furthermore, angiogenesis (collateral growth), a substantial element ofimprovement in flow-through, did not prolong walking time in patientsafflicted with limb ischemia when supporting GM-CSF treatment wasapplied without induction of microvessel growth and stabilization (vanRoyen et al., Circulation 2005, 112:1040-6). In contrast, adaptivecollateralization (Schierling et al., J Vasc Res 2009, 46:365-374)occurred when a proangiogenic factor like VEGF-A was combined with thematuration factors PDGF-B (Kupatt et al., J Am Coll Cardiol 2010,56:414-22) or angiopoietin-1 (Smith et al., J Am Coll Cardiol 2012,59:1320-8). On the other hand, inhibition of NF-κB signaling, hamperingVEGF-A and PDGF-B expression led to a hyper-branched and immaturecollateral network (Tirziu et al., Circulation 2012, 126:2589-600).Consequently, an increase in stable and regulated microvessels isnecessary for a successful induction of functional neovascularization.

Event-free survival of patients might be improved significantly usinggene therapy in cases of angiogenesis, arteriogenesis, in addition toimproved heart function. However, for these purposes, it is necessary toselect the correct gene therapy vector and target cells. The presentinvention advantageously solves these problems through the use of AAVvectors in vascular gene therapy strategies against coronary heartdisease.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to an adeno-associated viralvector (AAV vector) comprising a gene encoding a myocardin-relatedtranscription factor A (MRTF-A).

In another embodiment, the invention relates to an adeno-associatedviral vector (AAV vector) comprising a gene encoding a thymosin β4(Tβ4).

The AAV vector can be an AAV2/9 or an AAV vector pseudotyped withenvelope proteins of AAV9, preferably AAV2.9, AAV1.9, or AAV6.9.

In one embodiment, the AAV vector comprises a gene encoding an MRTF-A.

In one embodiment, the invention relates to an adeno-associated viralvector (AAV vector) comprising a gene encoding a myocardin-relatedtranscription factor A (MRTF-A), and a second gene encoding a thymosinβ4 (Tβ4) and/or a third gene encoding an MRTF-A.

In one embodiment, the first gene is under the control of acardio-specific promoter. In one embodiment, the first gene is under thecontrol of a CMV promoter, an MRC2 promoter, a MyoD promoter, or atroponin promoter.

Furthermore, the invention also relates to a pharmaceutical compositioncomprising an AAV vector of the invention and a pharmaceuticallyacceptable carrier.

The invention further relates to an AAV vector of the invention or apharmaceutical composition of the invention for use as a medicament. Inone embodiment, the AAV vector of the invention or the pharmaceuticalcomposition of the invention is for use in the treatment of coronaryheart disease or peripheral ischemia in a mammal, preferably in a human,a mouse, a rabbit, or a pig. The coronary heart disease can be an acuteheart attack, myocardial ischemia, stable angina pectoris, and/orhibernating myocardium.

In one embodiment, the mammal is a human No-Option-Patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Angiogenesis induced by MRTF activation and translocation intothe nucleus via CCN1 and CCN2 activation

FIG. 1a and FIG. 1b , MRTF-A transfection increased endothelial cellmigration in a wound scratch assay in vitro (bordered area=uncoveredarea); and FIG. 1c and FIG. 1d tubus formation of human microvascularendothelial cells (HMECs) in vitro (lpf=low power field). Overexpressionof Tβ4 showed similar effects if no MRTF shRNA was co-administered or aTβ4 mutant (Tβ4m) lacking the G actin binding motif KLKKTET was used(scale bar: 200 μm). FIG. 1e Tβ4 transfection of myocytic HL-1 cellsenabled translocation of MRTF-A (green fluorescence) into the nucleus(blue fluorescence), an effect which was absent if the Tβ4m constructwithout the G actin binding site was used (scale bar: 20 μm). FIG. IfTβ4 transfection of HL-1 cells induced an MRTF-SRF-sensitive luciferasereporter (comprising three copies of the SRF binding sitec-fos=p3DA.Luc, see Posern et al., Mol. Biol. Cell 2002, 13:4167-78), incontrast to transfection with the Tβ4 mutant. FIG. 5g Tβ4-induced tubusformation was abolished in the case of shRNA co-transfection of theMRTF-SRF target gene CCN1 (Cyr61) (scale bar: 200 μm). FIG. 1h and FIG.1i Tubus maturation, evaluated as pericyte recruiting (PC, greenfluorescence) on endothelial rings (EC rings, red fluorescence, scalebar: 200 μm), was induced by MRTF-A and Tβ4. Co-transfection of shRNAagainst the MRTF target gene CCN2 (CTGF) abolished the Tβ4 effect(mean±standard deviation, n=5, * p<0.05, ** p<0.001).

FIG. 2: The Tβ4-MRTF-A signaling cascade induces angiogenesis in vitro

FIG. 2a Tβ4 transfection of cardiomyocytic HL-1 cells enables thetranslocation of MRTF into the nucleus, an effect lacking when a Tβ4mconstruct without acting binding site was used. FIG. 2b Analysis of theMRTF-A protein level in the nucleus by Western blot showed an elevatedMRTF-A protein level after Tβ4 overexpression in HL-1 cells. Tβ4m didnot increase the MRTF-A level. FIG. 2c and FIG. 2d qRT-PCR shows thatCCN1/2 shRNA prevented accumulation of CCN1/2 transcripts after Tβ4expression. FIG. 2e and FIG. 2f rAAV.Tβ4-transduced cardiomyocytic HL-1cells induced angiogenesis (tubus formation) in endothelial cellsco-cultured with HL-1 cells, if no MRTF shRNA was co-transduced, whereasrAAV.Tβ4m had no effect (scale bar: 200 μm). FIG. 2g and FIG. 2h MRTF-AmRNA expression levels (g) and MRTF-A protein level (h) were notinfluenced by Tβ4 overexpression, but were significantly elevated afterMRTF-A transfection. FIG. 2i , FIG. 2j Tubus formation after Tβ4 releasefrom rAAV.Tβ4-transduced HL-1 cells was disrupted by CCN1 shRNA (scalebar: 200 μm). FIG. 2k and FIG. 2l Co-transfection of rAAV.Tβ4 and CCN2shRNA did not influence Tβ4-induced tubus formation. Furthermore, CCN1shRNA did not influence recruitment of pericyte-like cells(mean±standard deviation, n=5, * p<0.05, ** p<0.001).

FIG. 3: Importance of MRTF signaling for neovascularization in vivo

FIG. 3a qRT-PCR analysis showed an increase in MRTF-A in the ischemichind limb transduced with rAAV.MRTF-A. FIG. 3b rAAV.MRTF-A inducedMRTF/SRF target genes CCN1 and CCN2 in vivo. FIG. 3c and FIG. 3drAAV.MRTF-A transduction increased the capillary/muscle fiber ratio(c/mf) in a manner similar to MRTF activator Tβ4. rAAV.Tβ4m, a mutantwithout the G actin binding domain, or co-application of Tβ4 andrAAV.MRTF-A-shRNA, had no effect (PECAM-1 staining, scale bar 100 μm).FIG. 3e and FIG. 3f Functionally, transduction with rAAV.MRTF-A and-Tβ4, but not rAAV.Tβ4m or rAAV.Tβ4+MRTF-shRNA, improved the perfusionof the hind limb on day 3 and day 7. FIG. 3g After rAAV.Crevector-induced MRTF-B deletion in MRTF-A-deficient mice(Mrtfa^(−/−)/b^(flox/flox)+rAAV.Cre=MRTF-A/B^(−/−)Vi), Tβ4 transductioncould not induce angiogenesis, in contrast to Mrtfa^(+/−)/b^(flox/flox)mice (=MRTF-A/B^(+/−)). FIG. 3h Perfusion increased by rAAV.Tβ4 wassuppressed in MRTF-A/B^(−/−)Vi mice. FIG. 3i and FIG. 3j In CCN1^(−/−)Vimice (=CCN1^(flox/flox)+rAAV.Cre), both the increase of thecapillary/muscle fiber ratio (PECAM-1 staining, scale bar 100 μm) andthe increase of hind limb perfusion FIG. 3k and FIG. 3l were suppressed(mean±standard deviation, n=5, * p<0.05, ** p<0.001).

FIG. 4: MRTF-A induced vessel growth in mouse hind limb ischemia

FIG. 4a Protocol for mouse hind limb ischemia. Intramuscular (i.m.) rAAVadministration was performed on day −14 and the femoral artery wasligated on day 0. Subsequent laser Doppler flowthrough measurements(LDF) were performed on days 0, 3, and 7. FIG. 4b i.m. injection ofrAAV.Cre induced homogenous muscle transduction, shown by a change ofTomato fluorescence (red) to GFP fluorescence (green) in Tomato reportergene mice. FIG. 4c i.m. injection of rAAV.LacZ (3×10¹² virus particles)led to a homogenous transduction (blue staining) of the targeted hindlimb, but not of the opposite one. FIG. 4d qRT-PCR detection of Tβ4 inthe rAAV.Tβ4-transduced ischemic hind limbs, but not in therAAV.LacZ-transduced hind limbs. FIG. 4e HPLC analysis showed anincrease of Tβ4 protein concentration in the rAAV.Tβ4-transducedischemic hind limbs. FIG. 4f rAAV.Tβ4 induced MRTF target genes CCN1 andCCN2 in vivo. FIG. 4g Tβ4-induced maturation of capillaries (pericyteinvestment, NG2 staining) was suppressed in MRTF-A/B^(−/−)Vi hind limbs.FIG. 4h and FIG. 4i In both MRTF-A^(−/−)/B^(flox/flox) mice (MRTF-Aknockout) and MRTF-A^(+/−)/B^(−/−) Vi mice (MRTF-B knockout) rAAV.Tβ4transduction induced an increase of capillary density (h) and perfusion(i). However, the rAAV.Tβ4 effect was largest in wild type mice(MRTF-A^(+/−)/B^(flox/flox)). Furthermore, there was no significantdifference between MRTF-A and MRTF-B knockout mice (mean±standarddeviation, n=4, * p<0.05 vs. control).

FIG. 5: Tβ4/MRTF-A-induced microvessel maturation: essential role forcollateral growth and improved perfusion

FIG. 5a HPLC analysis showed a significant increase of Tβ4 protein afterrAAV.Tβ4 transduction of ischemic rabbit hind limbs, whereasrabbit-specific Tβ4-Ala remained unchanged. FIG. 5b , FIG. 5c and FIG.5d rAAV.MRTF-A or rAAV.Tβ4 administration increased capillary density(PECAM-1 staining) and pericyte investment (NG2 staining, scale bar: 50μm), both of which were abolished by co-application of angiopoietin 2(rAAV.Ang2). FIG. 5e and FIG. 5f Angiographies of ischemic hind limbs onday 35 showed an increased collateral formation in rAAV.MRTF-A- andrAAV.Tβ4-treated animals (arrows show site of excision of the femoralartery). Co-application of rAAV.Ang2 abolished this effect. FIG. 5grAAV.MRTF-A and rAAV.Tβ4 induced an increase of perfusion in ischemichind limbs, unless rAAV.Ang2 or L-NAME, which inhibits nitrogen oxideformation, were co-applied (mean±standard deviation, n=5, * p<0.05, **p<0.001).

FIG. 6: Tβ4-MRTF-A-induced vessel growth in rabbits

FIG. 6a Protocol of a model for rabbit hind limb ischemia (femoralartery excision). FIG. 6b Pβ-galactosidase staining 5 weeks after i.m.injection of rAAV.LacZ into the rabbit hind limb. FIG. 6c and FIG. 6dqRT-PCR of Tβ4 (c) and angiopoietin 2 (d), normalized to GAPDH, incontrol and treated animals (n=3). FIG. 6e and FIG. 6f Tβ4overexpression only in the lower limb (rAAV.Tβ4 LL) increased capillarydensity (PECAM-1 staining) in the lower limb, whereas Tβ4 transductiononly in the upper limb (rAAV.Tβ4 UL) did not influence thecapillarization in the lower limb (scale bar: 50 μm). FIG. 6g and FIG.6h Collateralization was increased in the rAAV.Tβ4 UL group, and to aneven greater extent in the rAAV.Tβ4 LL group, whereas perfusion FIG. 6iwas increased only in the rAAV.Tβ4 LL group and not in the rAAV.T14 ULgroup. FIG. 6j Compared to rAAV.Tβ4-transduced rabbits, theco-administration of L-NAME did not decrease the NG2/PECAM-1 ratio,indicating robust and balanced microvessel growth. In contrast, L-NAMEor rAAV.Ang2 treatment alone was not able to increase the NG2/PECAM-1ratio as strongly as rAAV.Tβ4. FIG. 6k and FIG. 6l Tβ4-dependentincrease of collateralization and perfusion was significantly reduced ifL-NAME was co-administered (mean±standard deviation, n=5, ** p<0.01).

FIG. 7: MRTF-A improves collateral formation and perfusion inhibernating myocardium of pigs

FIG. 7a -FIG. 7c In hibernating pig myocardium (see FIG. 8a ),rAAV.MRTF-A transduction and ubiquitous overexpression of Tβ4 (Tβ4tg,see FIG. 9) induced capillary sprouting (PECAM-1 staining, scale bar: 50μm) and pericyte investment (NG2 staining). FIG. 7d and FIG. 7eMoreover, collateral growth was detected in rAAV.MRTF-A-transducedhearts, similarly to Tβ4tg hearts. FIG. 7f The regional flow reserve,obtained by fast atrial stimulation (130 beats per minute), wasincreased in rAAV-MRTF-A-transduced and Tβ4-transgenic hearts. FIG. 7gRegional myocardium function, measured by subendocardial segmentshortening at rest and under atrial stimulation (130 and 150 beats perminute), showed improved functional reserve either by rAAV.MRTF-Atransduction or in Tβ4tg hearts. FIG. 7h The ejection fraction, aparameter of global myocardium function, was improved inrAAV.MRTF-A-transduced animals on day 56, compared with day 28.Constitutively overexpressing animals (Tβ4tg), however, showed no lossof function on day 28. FIG. 7i Mechanisms of MRTF-mediated therapeuticneovascularization: MRTF-A or Tβ4 transduction induces an increasedamount of MRTF-A not bound to G actin that interacts with SRF upontranslocation into the nucleus and induces e.g. CCN1 and CCN2 as targetgenes. CCN1 enables capillary growth (angiogenesis), whereas CCN2increases pericyte investment (vascular maturation). Together, thesemechanisms induce collateral growth in a nitrogen oxide-dependentmanner, leading to therapeutic neovascularization (mean±standarddeviation, n=5, * p<0.05, ** p<0.001).

FIG. 8: Functional efficiency of the Tβ4-MRTF-A axis in chronicallyischemic pig hearts

FIG. 8a Protocol of the pig model for hibernating myocardium. FIG. 8bRT-PCR-detection of MRTF-A and Tβ4 in control pigs compared withrAAV.Tβ4 and Tβ4-transgenic (Tβ4tg) pig hearts. FIG. 7c Examples of LacZstaining (blue) after rAAV.LacZ retroinfusion (5×10¹² virus particles)into the pig heart. FIG. 7d Before treatment (on day 28), retentionanalysis of fluorescent microbeads at rest showed a reduced blood flowin the ischemic area of rAAV.LacZ und rAAV.MRTF-A hearts, but not ofTβ4tg hearts, similarly to the flow reserve FIG. 7e at fast heart rate(130 bpm). FIG. 7f 4 weeks after treatment (on day 56), the regionalmyocardial blood flow in rAAV.MRTF-A und Tβ4tg animals improved. FIG. 7gFurthermore, the Rentrop score showed an increased collateralization onday 56 in rAAV.MRTF-A-transduced or Tβ4tg hearts. FIG. 7h Examples ofMRT analysis on day 56 for control (left) and rAAV.MRTF-A-treated pighearts. FIG. 7i The left ventricular end-diastolic pressure (LVEDP)increased in ischemic hearts from day 28 to day 56 if MRTF-A was notoverexpressed. Tβ4tg constitutively overexpressing Tβ4 showed no changefrom day 28 to day 56 (mean±standard deviation, n=5, * p<0.05, **p<0.001).

FIG. 9: Production of Tβ4-transgenic pigs

Fibroblasts of donor pigs were isolated and cultured. pCMV-Tβ4 wastransfected by electroporation and the cells were cultured for 14 days.After detection of stable transfection of Tβ4, a somatic nucleartransfer into pig oocytes was performed. Offspring were analyzed for Tβ4expression and fibroblasts of Tβ4-expressing animals were cultured andsubsequently used for a second somatic nuclear transfer. Aftergenotyping, animals of this generation were used for the pig model ofchronic ischemia.

FIG. 10: MRTFs are necessary for Tβ4-induced cardioprotection

FIG. 10a and FIG. 10b rAAV.Tβ4 induced capillary growth (PECAM-1staining) and FIG. 10c pericyte investment (NG2 staining, scale bar 50μm), unless co-administration of rAAV.MRTF-shRNA prevented bothprocesses. FIG. 10d and FIG. 10e Collateral growth was detected inrAAV.Tβ4-transduced animals, but not after co-administration ofrAAV.MRTF-shRNA. FIG. 10f Rentrop scores showed increasedcollateralization after rAAV.Tβ4 transduction, except in the case ofco-administration of MRTF-A shRNA. FIG. 10g Regional myocardial bloodflow at flow reserve (atrial stimulation 130/min) improved inrAAV.Tβ4-treated animals, but not in rAAV.Tβ4+MRTF-shRNA hearts. FIG.10h Analysis of the ejection fraction showed improved systolicmyocardium function in rAAV.Tβ4-transduced animals (day 56), as comparedwith day 28 (day of transduction). No improvement of the ejectionfraction was observed in rAAV.Tβ4+MRTF-shRNA-treated hearts. FIG. 10iMRT images of rAAV.Tβ4-transduced hearts without (left) or with (right)rAAV.MRTF-shRNA co-administration. FIG. 10j Regional myocardiumfunction, measured by subendocardial segment shortening at rest and atatrial stimulation (130 and 150 bpm) shows increased functional reserveafter rAAV.Tβ4 but not rAAV.Tβ4+MRTF-shRNA transduction (mean±standarddeviation, n=5, * p<0.05, ** p<0.001).

FIG. 11: Production and cardial phenotyping of INS^(C94Y)-transgenicpigs (diabetes mellitus type I)

FIG. 11a Process of producing the INS^(C94Y)-transgenic pigs. FIG. 11bBlood glucose levels of wild type and diabetic pigs. FIG. 11cFluorescence staining of endothelial cells (PECAM-1-positive cells, red)and pericytes (NG-2-positive cells, green). FIG. 11d Number ofendothelial cells in the myocardium of wild type and diabetic pigs. FIG.11e Left ventricular end-diastolic pressure in animals with diabetesmellitus type I and wild type animals.

FIG. 12: Characterization of the chronically ischemic myocardium modelwith cardiovascular risk factors

FIG. 12a Protocol of the pig model for hibernating myocardium withdiabetes mellitus type I or hypercholesterolemia. FIG. 12b Blood glucoseconcentration of the specific groups of animals over the duration of theexperiment: control wild type; wild type treated with rAAV.Tβ4; controlwith diabetes; diabetes treated with rAAV.Tβ4. FIG. 12 c and FIG. 12dSerum trigylceride and cholesterol levels in animals withhypercholesterolemia (fat rich diet) and normal diet.

FIG. 13: Influence of rAAV.Tβ4 application on angio- and arteriogenesisin animals with diabetes mellitus type I

FIG. 13a Fluorescence staining of endothelial cells (PECAM-1-positivecells, red) and pericytes (NG-2-positive cells, green) in hibernatingpig myocardium of diabetic and wild type animals. FIG. 13b and FIG. 13cNumber of endothelial cells and pericytes. FIG. 13d Number ofcollaterals formed. FIG. 13e Rentrop score.

FIG. 14: Functional efficiency of rAAV.Tβ4 application in animals withdiabetes mellitus type I

FIG. 14a and FIG. 14b Left ventricular end-diastolic pressure on days 28and 56 and its change between these time points. FIG. 14c and FIG. 14dEjection fraction on days 28 and 56 and its change between these timepoints.

FIG. 15: Influence of elevated cholesterol levels on Tβ4-mediated angio-and arteriogenesis

Number of FIG. 15a endothelial cells, FIG. 15b collaterals, and FIG. 15cRentrop score in the ischemic area of hypercholesterolemic control andrAAV-Tβ4-treated animals.

FIG. 16: Functional efficiency of rAAV.Tβ4 application in animals withhypercholesterolemia

FIG. 16a and FIG. 16b Left ventricular end-diastolic pressure on days 28and 56 and its change between these time points in hypercholesterolemiccontrol and rAAV-Tβ4-treated animals. FIG. 16c and FIG. 16d Ejectionfraction on days 28 and 56 and its change between these time points inhypercholesterolemic control and rAAV-Tβ4-treated animals. FIG. 16eRegional myocardium function, measured as subendocardial segmentshortening at rest and with increased heart rate (130 and 150 beats perminute).

FIG. 17: rAAV.Tβ4 and rAAV.MRTF-A pretreatment in a mouse model ofsepsis

FIG. 17a Protocol of the sepsis tests in mice. FIG. 17b Scoring schemefor the assessment of sepsis symptoms in mice and determination of thestop criteria. FIG. 17c Peripheral arterial blood pressure values after12 and 24 hours in animals with sepsis treated with different rAAV. FIG.17d Symptom scores of the animals with sepsis in the treatment groups.FIG. 17e Cumulated survival after LPS-induced sepsis.

FIG. 18: Role of MRTF-A and Tβ4 in vascular integrity during sepsis

FIG. 18a and FIG. 18b Histologic analyses of the endothelial cells(PECAM-1-positive cells) and the pericytes (NG-2-positive cells) in thehearts and the peripheral musculature of mice with sepsis. FIG. 18c andFIG. 18d Exemplary images and quantitative analysis of a permeabilitymeasurement by means of fluorescently labeled high molecular dextran 6hours after induction of sepsis.

DETAILED DESCRIPTION OF THE INVENTION

In our experiments (see Examples), we have found that the combination ofa long-acting vector and the overexpression of an effective vasoactivegrowth factor represents a therapeutic option for patients with chronicischemic diseases of skeletal or heart muscle tissue. The combination ofan adeno-associated vector and thymosin β4 (Tβ4) or MRTF-A transgene,respectively, leads to robust therapeutic vessel reformation in threespecies (mouse, rabbit, and pig). This therapeutic neovascularization inturn leads to a notably improved perfusion in the models of peripheralarterial obstruction disease and chronic myocardial ischemia. In themodel of chronic ischemic cardiomyopathy in pigs it leads additionallyto increased heart function. This specific effect can be achieved evenin large animals with additional cardiovascular risk factors (elevatedsugar or lipid levels).

A key feature of MRTF-A activation is translocation into the nucleusafter decrease of G actin levels and export from the nucleus when theamount of G actin increases (Miralles et al., Cell 2003, 113:329-42;Vartiainen et al., Science 2007, 316:1749-52). Enforced expression ofMRTF-A or Tβ4, a peptide activating MRTF-A by G actin binding (FIG. 1a-FIG. 1i ), initiates an orchestrated micro- and macrovascular growthresponse in the case of chronic ischemia of peripheral (FIG. 3a -FIG. 3l, FIG. 5a -FIG. 5g ) and heart muscle cells (FIG. 7a -FIG. 7i ).Consistent with these observations, chronic dysfunction of hibernatingpig myocardium was resolved both by direct MRTF-A activation and MRTF-Aactivation via Tβ4 (FIG. 7). The idea that MRTF-A-SRF signaling providesmyofilaments is of particular interest, since a loss of the actincytoskeleton is a hallmark of hibernating myocardium caused by chroniccoronary hypo-perfusion (Bito et al., Circ Res 2007, 100:229-37). Thus,MRTF-A is located at the interface of myocyte and vascular regenerationin hibernating myocardium. Tβ4, the most abundant G actin-bindingpeptide of the cytosol, can influence vascular growth by endothelialmigration and sprouting (Grant et al., J Cell Sci 1995, 108:3685-94:Smart et al., Nature 2007, 445:177-82). A substantial role of MRTF-A inTβ4 signaling has been shown in vitro and in vivo, since MRTF-A shRNAcould suppress endothelial migration and sprouting (FIG. 1b , FIG. 1d )and micro- and macrovascular growth (FIG. 3d , FIG. 3f ) and functionalimprovement of the heart (FIG. 10). Correspondingly,endothelium-specific deficiency in MRTFs caused incomplete formation ofthe primary vascular plexus in the developing retina (Weinl et al., JClin Invest 2013, 123:2193-206). Furthermore, MRF, the main target ofMRTF-A, has recently been identified as essential for the behavior ofapical cells in sprouting angiogenesis after VEGF-A stimulation (Francoet al., Development 2013, 2321-33: Andoh et al., J Biochem 2006,140:483-9). Nevertheless, VEGF-A leads to the growth of immature andunstable capillaries (Dor et al., EMBO J. 2002, 21:1939-47), in contrastto Tβ4-MRTF-A, thus indicating a difference in the signaling mechanismsfor these two vascular growth factors.

Collectively, our data demonstrate that activation of Tβ4-MRTF viaCCN1/CCN2 augments collateral blood flow in the ischemic heart and hindlimb via induction of CCN1/CCN2. At the cellular level this responseinvolves endothelial sprouting via CCN-1 (CYR61) and maturation, i.e.pericyte investment, via CCN2 (CTGF), resulting in a stable andfunctional vascular network that can carry collateral blood flow andimprove conductance. Pericyte investment is crucial here, since Ang-2,by virtue of disrupting pericyte investment (Ziegler et al., J ClinInvest 2013, 123:3436-45), abolished the positive effects exerted byTβ4-MRTF signaling (FIG. 3). This finding supports a central role ofvessel maturation and balanced vessel growth and paves the way for newtherapeutic avenues towards functional neovascularization.

Therefore, the invention comprises in a first embodiment anadeno-associated viral vector (AAV vector) comprising a first geneencoding a myocardin-related transcription factor A (MRTF-A). AAVvectors herein are particles displaying the envelope of anadeno-associated virus while comprising in their interior asingle-stranded DNA encoding a gene of interest. The gene of interestcan be introduced into a target cell by infection of the target cellwith an AAV vector.

The MRTF-A can be derived from a human, a mouse, a rabbit, a pig, or anyother mammal.

Particularly preferred is the use of an AAV vector comprising envelopeproteins, in particular the cap protein, of AAV9. AAV9 shows heartmuscle tropism and thus provides for homogenous and stable expression inthe heart muscle of a plurality of species. However, an AAV vectorpseudotyped with AAV9 may also be used. By this a vector is meantcomprising envelope proteins of AAV9, but otherwise expressing proteinsof another strain and also containing genomic elements, for exampleinternal terminal repeats (ITRs), from the other strain. For example,AAV2.9 is an AAV2 vector pseudotyped with envelope proteins of AAV9. Forthe present invention, AAV2.9, AAV1.9, and AAV6.9 are suitable aspseudotyped vectors. By using a heart muscle-tropic vector, it isensured that expression of MRTF-A occurs in the heart muscle, where itcan initiate therapeutic neovascularization.

Alternatively, an AAV vector with skeletal muscle tropism may also beused, in particular for the treatment of peripheral ischemia. Examplesare AAV6, AAV1, AAV9, or vectors pseudotyped with these strains.

The vector of the invention can further comprise additional expressiblegenes, e.g. an expression cassette for a thymosin β4 (Tβ4) or an MRTF-B.The Tβ4 can be derived from a human, a mouse, a rabbit, a pig, or anyother mammal. The MRTF-B can be derived from a human, a mouse, a rabbit,a pig, or any other mammal. Expression of these genes in the heartmuscle also supports therapeutic neovascularization in myocardialischemia.

The MRTF-A gene in the vector of the invention is preferably under thecontrol of a cardio-specific promoter, i.e. a promoter enablingexpression mainly in the heart muscle. Exemplary cardio-specificpromoters are the MLC2 promoter, the α myosin heavy chain promoter(α-MHC promoter) and the troponin I promoter (TnI promoter). However,other constitutive or inducible promoters may be used, e.g. a CMVpromoter or a MyoD promoter. The MRTF-A gene can also be under thecontrol of several promoters.

Methods for the production of AAV vectors for the transfer of specificgenes of interest are known in the state of the art (see e.g. Bell etal., J Clin Invest 2011, 121:2427-35). One method consists in the tripletransfection of a suitable producer cell line, e.g. U293, and subsequentpurification by cesium chloride gradient, as described in the section“Materials and methods” below. Here, the producer cells are transfectedwith three vectors: A first vector encodes the gene of interest, flankedby corresponding packaging signals; a second vector encodes thenecessary AAV proteins, in particular rep and cap; and a third vectorprovides the adenoviral helper functions without which no AAV particleproduction is possible.

In a further embodiment, the invention relates also to a pharmaceuticalcomposition comprising a vector of the invention and a pharmaceuticallyacceptable carrier. The pharmaceutical composition can be destined forevery administration known in the art. Compositions for intravenous orintramuscular injection are preferred. The pharmaceutical compositioncan additionally comprise salts, buffers, stabilizers, coloring agents,thickeners, flavors, etc.

The invention also relates to the AAV vector described herein or thepharmaceutical composition of the invention for use as a medicament. Inparticular, such use can occur in a mammal for treatment of coronaryheart diseases or peripheral ischemia. Preferred mammals are human, pig,rabbit and mouse.

The term “coronary heart disease” means a disease of the coronaryvessels of the heart. The coronary heart disease can be myocardialischemia, acute heart attack (myocardial infarction), stable anginapectoris and/or hibernating myocardium, but also cardiac arrhythmiaand/or heart insufficiency. “Peripheral ischemia” is an insufficientperfusion or a complete loss of perfusion of a tissue or organ outsideof the heart, while “myocardial ischemia” affects the heart muscleitself.

The vectors of the invention are particularly suitable for the treatmentof “no option” patients. In such patients, all interventional andsurgical therapeutic options are exhausted. Generally, slowing theprogression of the disease by drug therapy is attempted. This howevertargets lipid reduction and platelet inhibition, but notneovascularization. Therapeutic neovascularization can overcome thishurdle, if molecular signaling pathways leading to balancedneovascularization are used. MRTF-A and also Tβ4 are two molecules thatinduce this type of balanced neovascularization (capillaries,microvascular maturation, and collateral formation) in ischemic tissuewith concomitant lack of unwanted side effects.

Furthermore, vectors of the invention are particularly suitable for thetreatment of subjects bearing additional cardiovascular risk factors.Such risk factors include diabetes mellitus, in particular diabetesmellitus type I or type II. The risk factor may also be an elevatedconcentration of cholesterol in the blood (hypercholesterolemia) thatcan be caused by a diet characterized as fat-rich. The elevatedcholesterol concentration can be elevated LDL cholesterol concentrationor elevated HDL cholesterol concentration.

EXAMPLES Example 1: Induction of Hallmarks of Angiogenesis by MRTF-A InVitro

We have found (FIG. 1a and FIG. 1d ) that MRTF-A induced hallmarks ofangiogenesis, i.e. migration and tubus formation of cultured humanmicrovascular endothelial cells, to a comparable degree as Tβ4. Thepro-angiogenic effect of MRTF-A was found to be dependent on the G actinbinding motif of Tβ4, since mutation of this domain and abolition of Gactin binding eliminated the effect of Tβ4 on vascular growth, similarto an shRNA shown to disrupt transcription of MRTF-A and -B (MRTF shRNA;Leitner et al., J Cell Sci 2011, 124:4318-31). Consistent therewith, Tβ4increased MRTF-A translocation into the nucleus (FIG. 1e , FIG. 2a andFIG. 2b ), similar to the transcription of an MRTF/SRF-dependentreporter gene containing three SRF binding sites of the c-fos promoter(p3DA.Luc, FIG. 1f ; Geneste et al., J Cell Biol 2002, 157:831-8). BothMRTF-A and Tβ4 induced expression of genes involved in microvasculargrowth, in particular CCN1, mediating angiogenesis (Hanna et al., J.Biol. Chem. 2009, 284:23125-36), and CCN2, which is relevant for theattraction of 10T/2 pericyte-like cells (FIGS. 2c-g ; Hall-Glenn et al.,PLoS ONE 2012, 7:e30562). We observed that Tβ4 transfection did notaffect the MRTF-A content (FIG. 2h ), in contrast to MRTF-Atransfection. Consistently with CCN1/2 being downstream of MRTFs andrelevant for vessel formation, disruption by CCN1 shRNA preventedTβ4-induced tubus formation (FIG. 1g ), whereas CCN2 shRNA suspended theattachment of a murine pericyte-like cell line (C3H/10T1/2) toendothelial tubi in vitro (FIG. 1i and FIG. 1j ).

Example 2: Treatment of Hind Limb Ischemia in the Mouse with AAV-BasedMRTF-A Gene Therapy

In order to further demonstrate the relevance of MRTF-A signaling invivo, we employed a mouse model with hind limb ischemia. Intramuscularinjection of recombinant AAV vectors (rAAV, FIG. 4a and FIG. 4c )increased tissue concentration of target proteins in the treated limb(FIG. 3a ) and transcript levels of downstream mediators CCN1 and CCN2(FIG. 3b , FIG. 4d and FIG. 4f ). Consistent therewith, rAAV.MRTF-Ainduced capillary growth (FIG. 3c and FIG. 3d ) and increased perfusionon day 7 (FIG. 3e and FIG. 3). As an upstream activator, Tβ4 had asimilar effect on vascular growth and function (FIG. 2c -FIG. 2f ),unless the G actin binding motif was missing (Tβ4m) or anrAAV.MRTF-shRNA was co-administered. This vector encodes an shRNAdirected against both MRTF-A and MRTF-B and having the sequence5′-GAUCCCCGCAUGGAGCUGGUGGAGAAGAAUUCAAGAGAUUCUUCUCCACCAGCUCCAUGUUUUUGGAAA-3′ (SEQ ID NO: 1). In order tofurther examine the relevance of Tβ4-induced vascular growth, rAAV.Crewas administered to Mrtfa^(−/−)-Mrtf^(flox/flox) hind limbs to generateMRTF-A/B double insufficiency. In Cre-induced MRTF-A/B knockout mice,Tβ4 was not capable of stimulating capillary growth (FIG. 3g ) andpericyte recruitment (FIG. 4g and FIG. 4h ) and of improving perfusion(FIG. 3h , FIG. 4i ) on day 7 after induction of ischemia. Similarly,hind limbs did not show Tβ4-mediated increase of capillaries (FIG. 3iand FIG. 3j ) or perfusion (FIG. 3k and FIG. 3l ) on day 7, if rAAV.Crewas administered to CCN1^(flox/flox) mice. Consequently, MRTF-Atransduction or MRTF-A activation via Tβ4-mediated G actin sequestrationstimulates transcription of CCN1 to mediate functional vascularregeneration.

Example 3: Treatment of Hind Limb Ischemia in the Rabbit with AAV-BasedMRTF-A Gene Therapy

The mutual dependence of microvascular growth and arteriogenesis for themediation of regeneration of flowthrough was studied in a rabbit modelof ischemic hind limbs (FIG. 6a ), which is compatible with topicalseparation of the microvascular growth area (lower limb) and thecollateralization area (upper limb). Regional transduction of ischemiccalf muscle with MRTF-A or Tβ4 (FIG. 5a , FIG. 6b and FIG. 6d ) led tofunctional neovascularization, including CD31⁺ capillary sprouting (FIG.5b and FIG. 5c ), NG2⁺ pericyte investment (FIG. 5b and FIG. 5d ) andcollateral growth (FIG. 5e and FIG. 5f ). In particular, MRTF activationvia Tβ4 transduction of the hip region, though capable of inducingmoderate collateral growth, did not increase perfusion, whereas limitingMRTF-A activation via Tβ4 to the calf region was sufficient tosignificantly stimulate micro- and macrovascular growth and perfusion(FIG. 6e -FIG. 6i ). Detachment of microvascular pericytes by enforcedangiopoietin 2 expression (FIG. 5b and FIG. 5d ) abolished Tβ4-mediatedcollateralization and flowthrough improvement (FIG. 5e and FIG. 5g ).Furthermore, blocking of flow-induced vasodilation by oraladministration of L-NAME, a non-selective nitrogen oxide synthaseinhibitor, did not affect capillary growth and maturation (FIG. 6j ),but prevented formation of collaterals and increased perfusion (FIG. 6kand FIG. 6l ). Thus nitrogen oxide, subsequent to microvascular growthand maturation, appears to mediate collateral growth. This observationis supplemented by the finding that direct Tβ4 injection into the areaof collateral growth (upper limb) did not improve perfusion to the samedegree as distant injection of rAAV.Tβ4 into the lower limb, the area ofmicrovascular growth (FIG. 6e -FIG. 6i ). These findings indicate thatmicrovascular maturation and nitrogen oxide signaling are processes thatmust take place in the sequence of MRTF-A-mediated vascular growth toachieve a functional neovascularization.

Example 4: Treatment of Hibernating Myocardium in the Pig with AAV-BasedMRTF-A Gene Therapy

Although both peripheral and coronary arteries perfuse muscle tissue,permanent contraction activity is a unique feature of the heart muscle,which requires a continuous oxygen supply. A chronic drop in oxygensupply changes the cellular composition of living cardiomyocytes in theischemic area, leading to a regional loss of contraction force calledhibernating myocardium (Heusch and Schulz, J Mol Cell Cardiol 1996,28:2359-72; Nagueh et al., Circulation 1999, 100:490-6). Withincardiomyocytes, hallmarks of hibernating myocardium are reducedmyofilament (Bito et al., Circ Res 2007, 100:229-37) and mitochondriacontent and increased glycogen content (St. Louis et al., Ann ThoracicSurg 2000, 69:1351-7). We examined the potential of MRTF-A to resolvedysfunction in hibernating myocardium induced by percutaneousimplantation of a reduction stent in pig hearts (Kupat et al., J Am CollCardiol 2007, 49:1575-84) leading to a gradual occlusion of the ramuscircumflexus (RCx, FIG. 8a ). On day 28, following rAAV.MRTF-Aadministration into the ischemic area that significantly increasedMRTF-A content in the tissue (FIG. 8b ), we detected a significantlyhigher degree of capillary density and pericyte investment (FIG. 7a-FIG. 7c ). Collateral growth and perfusion under fast heart rate(130/min) were still impaired on day 28, i.e. before LacZ and MRTF-Atransduction (FIG. 8c -FIG. 8f ), but improved on day 56, i.e. 4 weeksafter MRTF-A transduction, but not after LacZ transduction (FIGS. 7d-f).

Increased collateral perfusion (FIG. 8g ) generated an improvedfunctional reserve of the ischemic area at fast heart rate (130 and 150beats per minute, FIG. 7g ). At the same time, we observed an improvedejection fraction as a marker of global systolic function (FIG. 7 h) anda drop of the left ventricular end-diastolic pressure (FIG. 8i ), apredictive marker of the beginning of heart failure.

Transgenic pigs that ubiquitously and constitutively express Tβ4 (FIG.9) showed similar capillary growth and maturation (FIG. 7a -FIG. 7c ).On day 56, the blood flow reserve in the ischemic area was increased(FIG. 7f ) and the functional reserve in the ischemic region (FIG. 7g )or the entire heart (FIG. 7h ) demonstrated an increase similar torAAV.MRTF-A-treated hearts. In particular, due to the constitutive Tβ4overexpression from day 0 to day 28, Tβ4tg animals did not experience asignificant loss of perfusion or myocardial function at rest or at fastheart rate (FIG. 7g , FIG. 8d , FIG. 8g , FIG. 8i ).

Furthermore, rAAV.Tβ4-induced micro- and macrovascular growth andsubsequent increases in the perfusion reserve were suppressed wheninhibiting MRTF-A shRNA was co-administered (FIG. 10a -FIG. 10f ). Theoverall gain in global (FIG. 10h , examples in FIG. 10i ) and regionalmyocardium function (FIG. 10j ) was abolished when Tβ4 transduction wascombined with MRTF-A inhibition by a suitable shRNA.

We therefore demonstrate, using a combined genetic and physiologicapproach in each of a mouse, rabbit and pig model, that MRTFs stimulatethe growth and maturation of microvessels as well as an increasedcollateral blood flow after arterial occlusion in hind limb and coronarynetworks. Mechanistically, we show that MRTF translocation downstream ofthymosin β4 co-activates SRF and induces CCN1/CCN2, thereby leading toincreased angiogenesis and recruitment of vascular smooth muscle cellsand formation of functional vessels that can carry collateral flow (FIG.7i ).

Example 5: Treatment of Hibernating Myocardium in Diabetic Pigs withAAV-Based Tβ4 Gene Therapy

Generation and Cardial Phenotyping of INS^(C94Y)-Transgenic Pigs(Diabetes Mellitus Type I)

The generation of transgenic pigs bearing the C94Y mutation in theinsulin gene (INS^(C94Y)) is shown in FIG. 11. This mutation is alsodepicted in Renner et al., Diabetes 2013, 62:1505-1511. The C94Ymutation leads to misfolding of the insulin protein in the β cells ofthe pancreas and an accumulation of the misfolded insulin in theendoplasmic reticulum (ER). ER stress leads to β cell apoptosis andthereby eventually to diabetes mellitus type I.

First, an INS^(C94Y) expression vector was introduced into pigfibroblasts by means of nucleotransfection. After selection of thefibroblasts, a first round of somatic nucleus transfer into oocytes wasperformed. Subsequently, the offspring were analyzed by Southern blotand the animals with elevated blood glucose levels and delayed growthwere used for renewed cloning (see Renner et al. 2013). These animalswere then used for subsequent testing at 3-4 months of age.

Once insulin treatment was stopped, the animals showed a markedlyelevated blood glucose level (FIG. 11c and FIG. 11d ). Analyses of theheart tissue for endothelial cells (PECAM-1-positive cells, red) and forpericytes (NG-2-positive cells, green) revealed a marked reduction ofthe endothelial cell and pericyte number even without additional stress.The analysis of the left ventricular end-diastolic pressure shows asignificant increase in animals with diabetes mellitus type I as a signof reduced global heart function already at an early stage (FIG. 11e ;mean±standard deviation; n=4, * p<0.05, **p<0.001).

FIG. 12 shows further effects of diabetes mellitus Type I or a fat-richdiet on the myocardium of pigs. FIG. 12 illustrates the experimentalprotocol of the pig model for hibernating myocardium with diabetes typeI or hypercholesterolemia. Compared with the control groups(wt±rAAV.Tβ4), the INS^(C94Y)-transgenic animals with diabetes mellitustype I (labeled as control tg and rAAV.Tβ4 tg) showed elevated bloodglucose levels for the entire assay period (FIG. 12b ). However, nodifference appeared between the rAAV.Tβ4-treated group and the controlgroup either for the wild type group or for the transgenic animals (FIG.12c and FIG. 12d ). An influence of Tβ4 or MRTF-A on the blood glucoselevel is not to be expected. In the animals with hypercholesterolemia,considerably elevated triglyceride and cholesterol levels appeared inthe serum after 9 weeks of ingesting a fat-rich diet (mean±standarddeviation; n=4, **p<0.001).

Effect of rAAV.Tβ4 Application in Animals with Diabetes Mellitus Type I

In hibernating pig myocardium, rAAV.Tβ4 transduction induces capillarysprouting (PECAM-1 staining, red) and pericyte recruitment (NG-2staining, green) in both groups (wild type and diabetes); FIG. 13a -FIG.13c . Moreover, considerable collateral growth was induced byoverexpression of Tβ4 via rAAV (FIG. 13d ), and a considerably betterfilling of the distal blood vessel could be measured by means of theRentrop score (FIG. 13e ). The effect could also be measured in bothgroups: wild type and diabetes (mean±standard deviation; n=4, * p<0.05,**p<0.001).

The left ventricular end-diastolic pressure, a parameter of globalmyocardium function, which showed an increase in the control animals ofboth groups from day 28 to day 56, was considerably reduced in theanimals with rAAV.Tβ4 transduction (FIG. 14a and FIG. 14b ). Theejection fraction, a further parameter of global myocardium function,showed a further decrease of values from day 28 to day 56 in controlanimals, whereas the value after Tβ4 overexpression considerablyimproved in both groups (wild type and diabetes) (FIG. 14c and FIG. 14d; mean±standard deviation; n=4, * p<0.05, **p<0.001).

Example 6: Effects of Tβ4 Gene Therapy on the Myocardium of Pigs withHypercholesterolemia

In control animals receiving 9 weeks of a fat-rich feeding, aconsiderable reduction of capillaries (PECAM-1-positive cells) appearedin the ischemic area (FIG. 15a ). With rAAV.Tβ4 application, thecapillaries (PECAM-1-positive cells) in the ischemic area could beconsiderably increased. With rAAV.Tβ4 transduction, the collateralgrowth could be increased even in animals having elevated cholesterollevels (FIG. 15b ). This also led to a better filling of the distalvessel section in the ischemic area, as shown by the Rentrop score (FIG.15c ; mean±standard deviation; n=4, * p<0.05, **p<0.001).

The left ventricular end-diastolic pressure, a parameter of globalmyocardium function, which was increasing in the control animals fromday 28 to day 56, was considerably reduced in the animals with rAAV.Tβ4transduction (FIG. 16a and FIG. 16b ). The ejection fraction, a furtherparameter of global myocardium function, showed a further decrease ofvalues from day 28 to day 56 in control animals, whereas the value afterTβ4 overexpression improved considerably (FIG. 16c and FIG. 16d ). Theregional myocardium function, measured as segment shortening at rest andunder increased heart rate (130 and 150 beats per minute) showed animproved functional reserve in animals with rAAV.Tβ4 therapy (FIG. 16e ;mean±standard deviation; n=4, * p<0.05, **p<0.001).

Example 7: Role of MRTF-A and Tβ4 in Vascular Integrity of Mice withSepsis

FIG. 17a illustrates the protocol of the sepsis experiments conducted inmice. Sepsis was induced 14 days after rAAV treatment (rAAV.MRTF-A orrAAV.Tβ4) by injection of LPS. At seven time points after sepsisinduction (12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h), an assessment ofthe symptoms was carried out by means of the table shown in FIG. 17b .The transduction of MRTF-A or Tβ4 via rAAV before induction of sepsisleads to increased peripheral arterial blood pressure values after 12and 24 hours (FIG. 17c ). In the course of up to 36 hours after sepsis,the rAAV.Tβ4- and rAAV.MRTF-A-treated animals show considerably lowersymptom scores as compared with the rAAV.LacZ-treated control animals(FIG. 17d ). The cumulative survival after LPS-induced sepsis isconsiderably improved by overexpression of Tβ4 and MRTF-A (FIG. 17e ;mean±standard deviation; n=7-15, * p<0.05, **p<0.001).

Histological analyses of endothelial cells (PECAM-1-positive cells) andpericytes (NG-2-positive cells) showed an elevated cell number in theheart and the peripheral muscles of animals treated with Tβ4 (FIG. 18aand FIG. 18b ) compared to control animals transduced with rAAV.LacZ.Exemplary images and a quantitative analysis of a permeabilitymeasurement by means of fluorescently labeled high molecular dextran 6hours after sepsis induction are shown in FIG. 18c and FIG. 18d . Here aconsiderably reduced leakage of the indicator was observed after Tβ4overexpression compared to the lacZ-transduced control animals(mean±standard deviation; n=4, * p<0.05, **p<0.001).

Materials and Methods

The experiments described in the examples were performed using thetechniques described in the following.

Reagents

All cell culture media and chemicals were purchased from SIGMA(Deisenhofen), if not indicated to the contrary. Contrast agentSolutrast 370 was supplied by Byk Gulden (Konstanz).

Adeno-Associated Viral Vectors

Recombinant vectors rAAV.MRTF-A, rAAV.Tβ4, r.AAV.Tβ4m, rAAV.LacZ,rAAV.Cre, and rAAV. MRTF-shRNA were produced by means of tripletransfection of U293 cells. One plasmid encoded the transgene undercontrol of a CMV promoter flanked by cis-acting internal terminalrepeats of AAV2. In the case of rAAV.MRTF-A, this was the plasmidpAAV-CMV-mMRTF-A (SEQ ID NO:16). However, a plasmid encoding humanMRTF-A may also be used, e.g. pAAV-CMV-hMRTF-A (SEQ ID NO: 17). A secondplasmid provided AAV2 rep and AAV9 cap in trans (Bish et al., Hum. GeneTher. 2008, 19:1359-68), while a third plasmid (delta F6) supplementedadenoviral helper functions. Cells were harvested 48 hours later andvectors purified using a cesium chloride gradient as describedpreviously (Lehrke et al., Cell Metab 2005, 1:297-308). Viral titerswere measured by real time PCR versus the polyA tail of the bGH of thevector (see primer sequences in Table 1). Trans and helper plasmids weresupplied by courtesy of James M. Wilson, University of Pennsylvania.

Cell Culture

SatisFection (TPP AG, Trasadingen, Switzerland) was used for thetransfection of human microvascular endothelial cells (HMECs), murineendothelial cells (bEnd.3), and the myocytic cell line HL-1 according tothe manufacturer's instructions. 100 μl serum- and antibiotic-free DMEMmedium were mixed with 3 μl of SatisFection transfection reagent.

In Vitro Tubus Formation and Co-Culturing Experiments

For the Matrigel experiments, HMECs were transfected with pcDNA, MRTF-A,Tβ4±MRTF-shRNA, Tβ4m (lacking the G actin binding motif KLKKTET;Bednarek et al., J. Biol. Chem. 2008, 283:1534-44), or Tβ4±CCN1-shRNA.Cells (8000 cells per well) were seeded on Matrigel (BD Matrigel™Basement Membrane Matrix, BD Biosciences, San Jose, USA) in basalendothelium growth media with a supplement of 5% fetal calf serum andimages were made after 18 h. The number of rings in the low power fieldwas quantified.

In co-culturing experiments, HL-1 cells were transduced withr.AAV.Tβ4±CCN1-shRNA, rAAV.MRTF-shRNA, or rAAV.Tβ4m (1×10⁶ AAV6particles per cell). HL-1 and HMECs embedded in Matrigel (8,000 perwell) were physically separated by a semipermeable membrane. After 18 h,the HL-1 cells were removed and ring formation in the low power fieldwas quantified.

CH3/10T1/2 pericyte cell attraction to murine endothelial cells (bEnd.3)was tested after transfection of the endothelial compartment with pcDNA,MRTF-A, or Tβ4±CCN2-shRNA by means of SatisFection (Agilent, Boblingen).Endothelial cells were stained with DiD (red, Vybrant®, LifeTechnologies) and seeded on Matrigel (12.000 cells per well). After 6 h,pericyte-like cells stained with DiO (Vybrant®, Life Technologies)(2,000 cells per well) were added and migration to the tubi was allowedfor 2 h. The co-culturing images were made by means of confocal lasermicroscopy (Carl Zeiss, Jena).

Migration Assay

HMECs were transfected as above with the indicated transgenes. 60,000cells were grown to confluence in wells with a strip-like insert (ibidiGmbH, Planegg). After 48 h, the nuclei were stained with Syto62. Thencells were fixed with 2% PFA, permeabilized, and incubated with ananti-MRTF-A antibody (Santa Cruz Biotech, Santa Cruz, USA) and asecondary antibody (Alexa 488-coupled, Invitrogen, Karlsruhe). Imageswere made by means of confocal laser microscopy (Carl Zeiss, Jena) andthe mean fluorescence intensity of the area of 100 nuclei, identifiedwith Syto62, were automatically evaluated using the LS5 image browser.

HPLC Analysis

Detection of Tβ4 was performed as described earlier (Huff et al., Ann.N. Y. Acad. Sci. 2007, 1112:451-7). Here, tissue samples were disruptedby adding 4 M perchloric acid with 1% thiodiethanol up to a finalconcentration of 0.4 M. Mixtures were homogenized, incubated for 30 minat 4° C. and centrifuged for 10 min at 20,000 g. The supernatant wasanalyzed using reverse phase chromatography. In rabbits, endogenous andexogenous Tβ4 were distinguished by detection of the rabbit-specificTβ4-Ala.

Luciferase Assay

To determine MRTF-dependent luciferase activity, HMECs and HL-1 cellswere transfected with p3DA.Luc (=a construct of a synthetic promoterhaving three copies of the c-fos SRF binding site and a Xenopus type 5actin TATA box plus a transcription start site inserted in pGL3; Posernet al., Mol. Biol. Cell 2002, 13; 4167-78), an SRF reporter gene, and930 ng of pcDNA, Tβ4 or Tβ4m. Comparable transfection efficiencies wereensured by co-transfection of 50 ng ptkRL (Renilla luciferase reporter).Pellets of cells were obtained and lysed, further purified bycentrifugation for 10 min at 4° C. and 13.000 rpm and used for thedetermination of firefly luciferase activity and Renilla luciferaseactivity. The ratio of firefly/Renilla luciferase activity wascalculated.

RNA Modulation and Detection

Real time PCR (RT-PCR) was conducted with SYBR Green dye (iQ SYBR GreenSupermix, Bio-Rad, Munchen) and measured on an iQ cycler (Bio-Rad,Minchen). The primers are listed in Table 1. Expression levels werenormalized to GAPDH and shown as multiples of the pcDNA controlsituation. The comparative 2 DDCt method was performed as describedearlier (Pfosser et al., Cardiovasc Res 2005, 65: 728-36).

Western Blot Analysis of MRTF-A

For the analysis of whole MRTF-A protein, cell culture and tissuesamples were homogenized in 1 ml lysis buffer containing 20 mM Tris, 1mM EDTA, 140 mM NaCl, 1% Nonidet P-40 (NP-40), 0.005 mg/ml leupeptin,0.01 mg/ml aprotinin, 1 mM PMSF, pH7.5. 60 μg whole protein extract wereseparated by polyacrylamide gel electrophoresis with 10% sodium dodecylsulfate (SDS-PAGE). After electrophoresis, the proteins wereelectrotransferred to a PVDF membrane (Millipore, Billerica, USA),blocked with 5% fat-free milk in PBS buffer containing 0.1% Tween 20 andincubated overnight at 4° C. with primary antibodies against MRTF-A(C-19; Santa Cruz Biotech, Santa Cruz, USA). After washing, the membranewas incubated with a secondary antibody (donkey anti-goat IgG,HRP-conjugated; Santa Cruz Biotech, Santa Cruz, USA) and developed witha chemiluminescence reagent (ECL; GE Healthcare, Buckinghamshire,England). For analysis of the MRTF-A protein content in the nucleus orthe cytosol, respectively, a separation with the Ne-Per® reagents forcytoplasmic and nucleus extraction (Thermo Scientific, Rockford, USA)was conducted according to the manufacturer's guidelines. Then a Westernblot analysis was carried out as described above. As a control protein,either α-tubulin (6A204; Santa Cruz Biotech, Santa Cruz, USA) or, forthe nucleus fraction, lamin B1 (ZL-5; Santa Cruz Biotech, Santa Cruz,USA) was used.

Animal Experiments

Animal care and all experimental procedures were carried out understrict adherence to the German and NIH animal guidelines and have beenapproved by the Animal Protection Commission of the Government of UpperBavaria (AZ 55.2-1-54-2531-26/09, 130/08, 140/07). All animalexperiments were conducted at the Walter Brendel Center for ExperimentalMedicine in Munich.

Mouse Hind Limb Ischemia

Unilateral hind limb ischemia of the right leg was performed in maleC56Bl mice of the same age (Charles River, Sulzfeld) and inMRTF-A^(+/−)/B^(flox/flox), MRTF-A^(−/−)/B^(flox/flox),MRTF-A^(+/−)/B^(−/−)Vi (=MRTF-A-^(+/−)/B^(flox/flox)+3×10¹² rAAV.cre),MRTF-A^(−/−)/B^(−/−)Vi (=MRTF-A-^(−/−)/B^(flox/flox)+3×10¹² rAAV.cre)(Weinl et al., J. Clin. Invest. 2013, 123:2193-226) and CCN1^(−/−)Vimice (=Cyr61^(flox/flox)+3×10¹² rAAV.Cre; produced in the laboratory ofRalf Adams at the Max Planck Institute for Molecular Biomedicine inMünster) as previously described (Limbourg et al., Nat. Protocols 2009,4:1737-48). Before induction of ischemia (day −14), 3×10¹² AAV9 virusparticles were administered intramuscularly to the right leg asdescribed (Qin et al., PLoS ONE 2013, 8:e61831). On day 0, the left legunderwent mock surgery, whereas in the right leg the femoral artery wasligated. The measurements of post-ischemic blood flow recovery wereconducted by means of laser Doppler flowthrough cytometry (MoorInstruments, Devon, England). Measurements were made directly before andafter surgery, on day 3, and on day 7. The results are given as theratio of right leg to left leg including subtraction of the backgroundtissue value. RT-PCR and HPLC analysis were carried out on day 5 afterinduction of ischemia; tissue was collected from treated and non-treatedlegs. Analyses of capillary density and vascular maturation were carriedout on day 7 in all groups by means of PECAM-1 (sc1506, Santa CruzBiotech, Santa Cruz, USA) and NG2 staining (inMRTF-A^(+/−)/B^(flox/flox) mice; Chemicon, Nürnberg) in frozen tissuesamples of the M. gastrocnemius and M. adductor.

Rabbit Hind Limb Ischemia

On day 0, the complete femoral artery of the right leg in New Zealandrabbits was removed (Pfosser et al., Cardiovasc. Res. 2005, 65:728-736)and rAAV administration (5×10¹² virus particles) was performed by meansof intramuscular injection into the right hind limb as indicated. On day7 and day 35, angiography was performed by injection of contrast agent(Solutrast 370, Byk Gulden, Konstanz) into the ischemic leg with anautomatic injector (Harvard Apparatus, Freiburg). Furthermore,fluorescent microbeads (15 μm, Molecular Probes®, Life Technologies,Carlsbad, USA) were used for blood flow measurements in ischemic andnon-ischemic tissue. For blood flow analysis, tissue samples weredigested as previously described (Thein et al., Comput. Methods ProgramsBiomed. 2000, 61:11-21; Kupaxtt et al., J Am Coll Cardiol 2010,56:414-22). Fluorescence analysis was carried out with a Tecan Saphire 2microtiter plate reader at the emission wavelengths 680 nm, 638 nm, 598nm, 545 nm, 515 nm, 468 nm, and 424 nm, depending on the fluorescent dyeemployed. Calculations were carried out as described previously (Lebherzet al., Endothelium 2003, 10:257-65). Analysis of capillary density andvascular maturation was carried out by means of PECAM-1 (sc1506, SantaCruz Biotech, Santa Cruz, USA) and NG2 staining (inMRTF-A^(+/−)/B^(flox/flox) mice; Chemicon, Nürnberg) in frozen tissuesamples of the ischemic and non-ischemic leg.

Chronic Myocardial Ischemia in Pigs

Pigs were anesthetized and treated as described previously (vonDegenfeld et al., J. Am. Coil. Cardiol. 2003, 42:1120-8). To this end, areduction stent coated with a PTFE membrane was implanted in theproximal RCx, leading to 75% reduction of blood flow. Correctlocalization of the stent and permissibility of the distal vessel wereensured by the injection of contrast agent. On day 28, the baselinemeasurements for global myocardium function (left ventricularend-diastolic pressure=LVEDP, ejection fraction=EF) and myocardialperfusion (fluorescent microbeads, 15 μm, Molecular Probes®) wereconducted. Then selective pressure-regulated retroinfusion into thelarge cardiac vein draining the RCx-perfused myocardium was carried outfor κ×10¹² virus particles of rAAV.MRTF-A and rAAV.Tβ4±rAAV.MRTFA-shRNA.On day 56, the measurements of global myocardium function and blood flowwere repeated and the regional myocardium function of the ischemic andnon-ischemic area were determined (at rest and under fast heartstimulation, 130 and 150 bpm). Post mortem angiography was carried outfor the calculation of the collateral value and analysis by Rentropscore (0=no filling, 1=side branch filling; 2=partial main vesselfilling; 3=complete main vessel filling). Tissue was collected for theanalysis of regional myocardial blood flow and immunohistology.

Global Myocardial Function

On day 28 and day 56, the global myocardial function (LVEDP) wasexamined by a Millar pressure tip catheter (Sonometrics, Ontario,Canada). An angiogram of the left ventricle for global myocardialfunction was performed on day 28 and day 56. The ejection fraction wasobtained by planimetry of the end-systolic and end-diastolic angiogramimages (Image J 1.43u, National Institute of Health, USA).

Regional Myocardial Function

On day 56 after induction of ischemia, sternotomy was performed andultrasound crystals were placed subendocardially in a standardizedmanner in the non-ischemic area (LAD control region) and the ischemicarea (Cx perfused region). Subendocardial segment shortening (SES,Sonometrics, Ontario, Canada) was examined at rest and under elevatedheart rate (functional reserve, rate 130 and 150) and evaluated off-linedepending on ECG.

Regional Myocardial Blood Flow

The analysis of regional myocardial blood flow was performed on day 28(before rAAV treatment) and day 56 (28 days after AAV treatment) bymeans of fluorescent microbeads (Molecular Probes®). The microbeads (15μm, 5×10⁶ particles per injection) were injected into the left ventriclewith a pigtail catheter. Blood flow measurements were carried out atrest and at elevated heart rate (130 bpm). The fluorescence content wasanalyzed by means of a Tecan Sapphire 2 microtiter plate reader and acalculation of the regional myocardial blood flow was performed, eitheras ml/g tissue absolute or as the ratio to the non-ischemic region atrest (% non-ischemic blood flow; Kupatt et al., J Am Coil Cardiol 2010,56:414-22).

Histology

Tissue samples of the ischemic and non-ischemic area were examined forcapillary density (PECAM-1-positive cells, red) and pericyte investment(NG-2-positive cells, green). Staining of capillaries was carried outwith an anti-CD31 antibody (SC1506, Santa Cruz Biotech, Santa Cruz, USA)and a rhodamine-labeled secondary antibody, while vascular maturationwas quantified by pericyte co-staining (anti-NG2-antibody AB5320,Millipore, Billerica, USA). Images of the ischemic and non-ischemicregion were made with high power field magnification (40 times), and 5independent images per region (ischemic and non-ischemic) and animalwere quantified.

rAAV Transduction Efficiency

For the evaluation of the rAAv transduction efficiency, control mice,rabbits and pigs were treated with rAAV.LacZ. Cryostatic sections of theLacZ-transduced animals were prepared and stained for β-galactosidase(blue staining). Furthermore, RT-PCR for the several transgenes wascarried out using the primers described in Table 1 and analyzed asdescribed above.

Tomato Reporter Gene Mice

These mice homozygously expressing mT/mG (Jackson Laboratory, BarHarbor, USA) express loxP sites on both sides of a membrane-directedtdTomato (mT) and a membrane-directed eGFP (Muzumdar et al., Genesis2007, 45:593-605). Cre expression via rAAV.Cre for the determination ofvirus transduction efficiency deleted mT (red fluorescence) in the cellsand enabled eGFP expression (green fluorescence) in the same cells (FIG.4b ).

Statistical Methods

The results are shown as means±standard deviation. Statistical analyseswere performed using one-way variance analysis (ANOVA). Every time asignificant effect was found (p<0.05), we conducted multiple comparativetests between groups with the Student Newman Keul method (IBM SPSS 19.0;IBM, Chicago, USA). Differences between groups were regarded assignificant at p<0.05.

TABLE 1 Primer sequences used for PCR: BGH forward5′-TCT AGT TGC CAG CCA TCT GTT GT-3′ SEQ ID NO: 2 BGH reverse5′-TGG GAG TGG CAC CTT CCA-3′ SEQ ID NO: 3 GAPDH forward5′-AAT TCA ACG GCA CAG TCA AG-3′ SEQ ID NO: 4 GAPDH reverse5′-ATG GTG GTG AAG ACA CCA GT-3′ SEQ ID NO: 5 Tβ4 forward5′-TCA TCG ATA TGT CTG ACA AAC-3′ SEQ ID NO: 6 Tβ4 reverse5′-CAG CTT GCT TCT CTT GTT CAA-3′ SEQ ID NO: 7 MRTF-A forward5′-AAT CCA TGG GTC GAC GGT ATC GAT-3′ SEQ ID NO: 8 MRTF-A reverse5′-ATA CCA TGG TCA GGC ACC GGG CTT-3′ SEQ ID NO: 9 CCN1 (CYR61) forward5′-GCT AAA CAA CTC AAC GAG GA-3′ SEQ ID NO: 10 CCN1 (CYR61) reverse5′-GGC TGC AAC TGC GCT CCT CTG-3′ SEQ ID NO: 11 CCN2 (CTGF) forward5′-CCC TAG CTG CCT ACC GAC T-3′ SEQ ID NO: 12 CCN2 (CTGF) reverse5′-CAT TCC ACA GGT CTT AGA ACA GG-3′ SEQ ID NO: 13 Ang2 forward5′-TCG AAT ACG ATG ACT CGG TG-3′ SEQ ID NO: 14 Ang2 reverse5′-GTT TGT CCC TAT TTC TAT C-3′ SEQ ID NO: 15

1. An adeno-associated viral vector (AAV vector) comprising a geneencoding a neovasoactive growth factor, wherein the neovasoactive growthfactor is a myocardin-related transcription factor A (MRTF-A) orthymosin β4 (Tβ4), or a combination thereof.
 2. The AAV vector accordingto claim 1, wherein the AAV vector is an AAV9 vector or an AAV vectorpseudotyped with AAV9 envelope proteins selected from AAV2.9, AAV1.9 andAAV6.9.
 3. The AAV vector according to claim 1, further comprising agene encoding an MRTF-B.
 4. The AAV vector according to claim 1, whereinthe MRTF-A gene is under the control of a cardio-specific promoter. 5.The AAV vector according to claim 4, wherein the cardio-specificpromoter is a CMV promoter, an MRC2 promoter, a MyoD promoter, or atroponin promoter.
 6. A pharmaceutical composition comprising an AAVvector of claim 1 and a pharmaceutically acceptable carrier.
 7. The AAVvector according to claim 1 formulated for treating coronary heartdisease or chronic ischemic diseases in a mammal, wherein the AAV vectoris present in an amount effective to enhance MRTF-A activation.
 8. Amethod of treating coronary heart disease or peripheral ischemia in amammal by administering the pharmaceutical composition of claim
 6. 9.The method according to claim 8, wherein the coronary heart disease isacute heart attack, myocardial ischemia, stable angina pectoris and/orhibernating myocardium.
 10. (canceled)
 11. The method according to claim8, wherein the mammal is suffering from diabetes mellitus orhypercholesterolemia.
 12. The method of claim 8, wherein the mammal is ahuman, a mouse, a rabbit, or a pig.
 13. The method according to claim12, wherein the human is a human no option patient.
 14. The AAV vectoraccording to claim 7, wherein the coronary heart disease is acute heartattack, myocardial ischemia, stable angina pectoris and/or hibernatingmyocardium.
 15. The AAV vector according to claim 7, wherein the mammalis a human, a mouse, a rabbit, or a pig.
 16. The method according toclaim 15, wherein the human is a human no option patient.
 17. The AAVvector according to claim 7, wherein the mammal is suffering fromdiabetes mellitus or hypercholesterolemia.
 18. A method for therapeuticvessel reformation and increasing vessel profusion comprisingadministering the AAV vector of claim 1 to a mammal in an amountsufficient to enhance MRTF-A activation.
 19. The method according toclaim 18, wherein the mammal is a human, a mouse, a rabbit, or a pig.20. The method according to claim 18, wherein the mammal suffers fromcoronary heart disease, chronic ischemic diseases, diabetes orhypercholesterolemia.